Molecular cloning of pig Rad51, Rad52, and Rad54 genes, which are involved in homologous recombination machinery.
BACKGROUND
The low rate of homologous recombination in somatic cells is considered to be an urgent issue. Therefore, we molecularly cloned three genes that relate to efficient homologous recombination.


METHODS
Polymerase chain reaction (PCR) was performed to isolate candidate cDNA fragments from a pig spleen cDNA library with the corresponding primer sets deduced from multiple alignment analysis of other mammalian genes. A 5'- and 3'-RACE PCR experiment was performed to determine the complete cDNA sequences.


RESULTS
The complete cDNA sequences of the pig RAD51, RAD52, and RAD54 genes, which are closely related to homologous recombination events, were identified using molecular cloning technique. The cDNA sequences of three genes were successfully isolated by PCR-based methods. As a result, we determined the sequences of pig RAD51 (1663 bp, 339 aa), RAD52 (1884 bp, 406 aa), and RAD54 (2884 bp, 747 aa). The nucleic acid sequence homologies of the pig RAD51, RAD52, and RAD54 genes compared with the corresponding human genes were 92.9%, 77.3%, and 90.0%; the corresponding amino acid sequence homologies were 98.8%, 71.1%, and 95.0%, respectively.


CONCLUSIONS
The knockout of alpha-1,3-galactosyltransferase in pigs resulted in a drastic reduction in xenoantigenicity. However, other xenoantigens, in particular, the non-Gal antigens, also need to be down-regulated. Gene transfer to alter expression levels of these recombination-related molecules and/or ex ante evaluation of expression profiles of these genes in primary cultures of somatic cells constitute a new approach to enhancing homologous recombination events during the production of gene knockout pigs.
Matsunami
K
K
Division of Organ Transplantation, Department of Molecular therapeutics, Osaka University Graduate School of Medicine, Osaka, Japan.
Otsuka
H
H
Xu
H
H
Firdawes
S
S
Yamamoto
A
A
Ishimaru
A
A
Fukuzawa
M
M
Miyagawa
S
S
D000251:Adenosine Triphosphatases / Q000737:chemistry / Q000235:genetics*; D000595:Amino Acid Sequence; D000818:Animals; D030801:Animals, Genetically Modified; D018076:DNA, Complementary / Q000737:chemistry / Q000235:genetics; D017353:Gene Deletion; D008969:Molecular Sequence Data; D051135:Rad51 Recombinase / Q000737:chemistry / Q000235:genetics*; D051721:Rad52 DNA Repair and Recombination Protein / Q000737:chemistry / Q000235:genetics*; D011995:Recombination, Genetic; D013552:Swine
D018076:DNA, Complementary; D051721:Rad52 DNA Repair and Recombination Protein; D051135:Rad51 Recombinase; D000251:Adenosine Triphosphatases